Our objective will be to establish synchronous animal cell culture systems in which the existence of a limit cycle oscillator can be tested, and to further characterize the mechanisms by which the onset of cell division, the initiation of DNA synthesis and the expression of enzyme activity maxima are timed. During the tenure of this grant the experimental work will concentrate on the phase response of animal cells to serum pulses, to temperature shifts and to various drugs, and on the ability of cells arrested by hydroxyurea or isoleucine deprivation to undergo subthreshold oscillations. The clock in animal cells has no clearly recognized time cues. To further our understanding of the cellular timekeeping system we will need to identify the time cues employed by the cell, or by the organism to entrain the cell. In the circadian system the time cues are light and temperature and we can only assume that in the cells of a metazoan organism light, as a cue, would be mediated by a hormonal factor, and ultimately by some internal second messengers such as cAMP or changes in ion flux. We will begin to look among the known serum factors for one which fits the criteria of a zeitgeber and to develop cell culture systems in which the clock can be discerned more clearly.